Reconstitution of SNAREs

The reconstitution of the synaptobrevin or the ΔN49-complex into lipid vesicles was performed as previously described by Schwenen et al.²⁴ Therefore lipid films (630 nmol) were dissolved in HEP150/HEP220-buffer (50 µL) containing n-octyl-β-D-glucopyranoside (NOG) (100 mM) and incubated for 30 minutes to receive a solution of micelles. Both stock solutions of the proteins contained 1% 3-[(3-Chloamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The detergent molecule were added to the lipids in a small buffer solution to generate micelles. The critical micelle concentration (CMC) has to be considered as a crucial value for the use of detergents. At this critical concentration either detergent molecules form micelles as well as the lipids in the solution with the detergent are dissolved to form mixed micelles.⁹⁷ The micelles and proteins were mixed and incubated for 30 minutes on ice. GUVs were prepared with the ΔN49-complex (2 nM), LUVs with synaptobrevin (2 nM). To remove the detergent molecules a Sephadex column (illustra NAP-25, GE Healthcare) was prepared with HEP150/HEP220–buffer (Figure 3.13). After the elution of the micelle-protein mixture the resulting SNARE protein containing LUVs (50-900 nm in diameter) were collected in a reaction tube. The size distribution of the LUVs was measured by dynamic light scattering (chapter 3.4.1). Concentrating the vesicle solution in a vacuum centrifuge (Concentrator 5301, Eppendorf, Hamburg) to a volume of 80 to 150 µL and elution in a column with pure water results in an ion free vesicle solution that was again concentrated to a final volume of approximately 100 µL. The vesicles solution with synaptobrevin was given into a small reaction tube and dried in a desiccator filled with a saturated sodium chloride solution.

LUVs were obtained by dissolving the lipid film in the reaction tube for 30 minutes with HEP150/HEP220-buffer. Small droplets (2 µL) with LUVs containing the ΔN49-complex were given onto ITO-slides and dried in the desiccator. With the electroformation technique GUVs were produced (chapter3.1.3). The chamber inside was filled with sucrose solution (150 mOsmol/kg) and a sinusoidal voltage of 1.6 Vpp with a frequency of 12 Hz was applied for 2.5-3 h to yield GUVs with a size of about 3-50 µm in diameter.

The GUV- and LUV-solution can be stored on ice for a maximum of one day. The size distribution of the LUVs diameter was determined by dynamic light scattering.

Figure 3.13. Scheme of SNARE-reconstitution into the vesicle membranes by the detergent dilution method. The detergent solution containing the lipids and SNARE proteins was given onto the Sephadex column. Elution with buffer HEP150/HEP220 results in a LUV solution that was collected in a reaction tube. While elution of the detergent, the SNARE proteins incorporate into the lipid bilayer of the LUVs by inserting their trans membrane sequence in the amino acid chain.

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3.4.1. Dynamic light scattering ‒ LUV size distribution

Dynamic light scattering (DLS) is a common technique to determine the size distribution of particles in hydrodynamic systems.^98-99 After the reconstitution of SNAREs with the detergent dilution chromatography the vesicle size distribution of the LUVs was determined by DLS. DLS measurements were performed using Zetasizer Nano S. A small volume of the LUV solution (100-200 µL) was given into a cuvette. Then the measurement was performed after an equilibration time of 120 seconds. Three measurements using the same sample were done and the resulting data averaged.

The measurement of the LUV size distribution is shown in figure 3.14. LUVs for that measurement went through the detergent dilution chromatography two times as described in chapter 3.4. The extrusion of the LUV solution was done with a 1 µm porous membrane to remove larger dust particles that could disturb the DLS measurement. Figure 3.14 shows that the LUV size distribution has a maximal intensity at around 200 nm in diameter. The size distribution of LUVs ranges from 50 nm up to 1 µm in diameter.

The measured hydrodynamic diameter is assumed to represent the actual diameter of the LUVs. Small LUVs with a diameter <300 nm cannot be detected with a confocal microscope because of ABBE’s resolution criterion but larger LUVs (>300 nm) can. This DLS measurement of LUVs serves as a fundamental information for the membrane fusion experiments to compare the size and volume of the GUVs and LUVs.

Figure 3.14. Measured size distribution of LUVs after the detergent dilution chromatography. LUV diameter is shown on a logarithmic scale.

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3.4.2. Vesicle Fusion assay after protein reconstitution

Figure 3.15. Scheme of the vesicle fusion assay used to test the successful reconstitution of SNAREs into the LUV membranes.

With the vesicle fusion assay, illustrated in figure 3.15, the reconstitution of the ΔN49-complex and the Syb was proved by mixing two fractions of LUVs each with one of the SNAREs and a fluorescently labeled lipid that was the corresponding FRET pair. LUVs were obtained after the detergent dilution to reconstitute the SNARE proteins into the membranes. At first, the LUV solution containing the dye A594 (50 µL) and the ΔN49-complex was added into a glass cuvette (108F QS, Hellma Analytics, Müllheim, Germany) with HEP220-buffer (800 µL). The second LUV fraction (50 µL) containing A488 and Syb2 was added to the buffer solution with the first LUV fraction and stirred for 45 minutes. While the addition of the two LUV solutions and stirring of the buffer solution the fluorescence intensity of A594 was measured at a wavelength of 627 nm with a fluorimeter (FluoroMax, Horiba, Kyoto, Japan). For this purpose an excitation lamp with a wavelength of 501 nm was used to excite the lipid dye A488 in the LUV membranes. Via the FRET-effect A594 shows a fluorescence if the membranes between the two LUV fractions fuse with each other. An increase in fluorescence intensity of A594 indicates LUV fusion between the LUV fractions with the SNARE proteins.

In figure 3.16 the fluorescence intensity of A594 at a wavelength of 627 nm is drawn in the graph. The red curve for LUVs containing SNAREs shows an increase in fluorescence intensity after the addition of the second LUV fraction containing A488 and Syb due to the fusion of the LUVs. As a reference, the blue curve in the graph, representing LUVs without SNAREs, shows only a slightly fluorescence intensity increase. With this vesicle fusion assay it was proven that fusion of membranes, LUV respectively, can be achieved with the used lipid composition and the reconstituted SNARE proteins.

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Figure 3.16. The vesicle fusion assay showed that membrane fusion occurs after the addition of the second LUV fraction (red) compared to LUVs without SNAREs (blue).

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